Human Bone Morphogenic Protein 7 (hBMP-7) is a member of the TGF-β superfamily of proteins and has recognized therapeutic potential as both a modulator of bone structure and renal function (Klahr, J. Nephrol. 16:179-185 (2003)). For example, active hBMP-7 could be used as a protein therapeutic in indications such as myelofibrosis, idiopathic pulmonary fibrosis, renal osteodystrophy, renal fibrosis, diabetic nephropathy, chronic obstructive pulmonary disorder (COPD), and osteoarthritis.
hBMP-7 is transcribed as mRNA containing a 1293 base pair open reading frame (the corresponding cDNA is shown at SEQ ID NO: 1) which is translated as a precursor protein of 431 amino acid residues (FIG. 1; SEQ ID NO: 2) that is proteolytically processed. The hBMP-7 precursor protein contains a signal peptide spanning residues 1 to 29 of SEQ ID NO: 2, a prodomain spanning residues 30-292 of SEQ ID NO: 2, and a mature form domain spanning residues 293-431 of SEQ ID NO: 2 (FIG. 1).
Proteolytic processing of the hBMP-7 precursor protein is believed to occur in several steps. First, the signal peptide directs hBMP-7 to the endoplasmic reticulum of the cell where folding occurs and hBMP-7 forms a homodimeric protein complex. Next, the hBMP-7 prodomain is removed by proteolytic processing to produce a covalently linked homodimer consisting of two antiparallel hBMP-7 mature domain peptide chains. This final homodimeric protein complex is the biologically active form and secreted by cells.
Secreted hBMP-7 interacts with surface receptors on other cells and soluble antagonists, such as ActRII, BMPR1a, and Noggin to mediate its biological effects and activity. Structural information concerning these interactions can be derived from a number of different BMP-7 crystal structures (Greenwald et al., Mol. Cell. 11:605-017 (2003); Griffith et al., PNAS. 93:878-883 (1996); Keller et al., Nat. Struct. Mol. Biol. 5:481-488 (2004); Groppe et al., Nature. 420(6916):636-642 (2002).
Although hBMP-7 (formerly known as osteogenic protein 1 (OP-1) by Ozkaynak et al. (EMBO J. 9(7), 2085-2093 (1990)) has been known at least since 1990, to date several unresolved problems have prevented its development as a therapeutic protein. First, recombinant expression of hBMP-7 in mammalian cells is extremely low relative to other proteins of similar size. This is incompatible with the large-scale commercial production and purification needed for therapeutic use of hBMP-7. Second, proteolytic processing of the mature domain of hBMP-7 can occur at any of four different sites in that domain. This results in a number of different mature forms of hBMP-7 being produced during the recombinant expression of this protein. This high degree of heterogeneity in the expressed protein is highly undesirable in a protein therapeutic. Third, the mature form of hBMP-7 has poor solubility at pH values near 7.0; thus, acidic pH values are required to maintain hBMP-7 solubility. However, acidic pH values are incompatible with most common delivery methods, such as intravenous injection, used to administer protein therapeutics to patients. Lastly, the prodomain of hBMP-7 associates non-covalently with the mature domain of hBMP-7. Thus, purifying the prodomain away from the mature domain of hBMP-7 is relatively difficult and is incompatible with large-scale commercial production and purification.
Thus, a need exists for hBMP-7 variant peptide chains with improved properties that are suitable for use as therapeutic proteins and are compatible with large-scale commercial production and purification.